Cd19 specific chimeric antigen receptor and uses thereof

ABSTRACT

The present invention relates to chimeric antigen receptors (CAR). CARs are able to redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-binding domain properties. In particular, the present invention relates to a Chimeric Antigen Receptor in which extracellular ligand binding is a scFV derived from a CD19 monoclonal antibody, preferably 4G7. The present invention also relates to polynucleotides, vectors encoding said CAR and isolated cells expressing said CAR at their surface. The present invention also relates to methods for engineering immune cells expressing 4G7-CAR at their surface which confers a prolonged “activated” state on the transduced cell. The present invention is particularly useful for the treatment of B-cells lymphomas and leukemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/891,296, filed Nov. 13, 2015, which is a National Stage entry ofInternational Application No. PCT/EP2014/059662, filed May 12, 2014,which claims benefit of U.S. Provisional Application No. 61/888,259,filed Oct. 8, 2013, and which is a continuation-in-part of U.S. patentapplication Ser. No. 13/892,805, filed May 13, 2013, acontinuation-in-part of International Application No. PCT/US2013/040755,filed May 13, 2013, and a continuation-in-part of InternationalApplication No. PCT/US2013/040766, filed May 13, 2013. The disclosuresof the prior applications are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ALGN_001_05US_SeqList_ST25.txt. The text file is˜52.6 kilobytes, was created on Nov. 13, 2020, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates to chimeric antigen receptors (CAR). CARsare able to redirect immune cell specificity and reactivity toward aselected target exploiting the ligand-binding domain properties. Inparticular, the present invention relates to a Chimeric Antigen Receptorin which extracellular ligand binding is a scFV derived from a CD19monoclonal antibody, preferably 4G7. The present invention also relatesto polynucleotides, vectors encoding said CAR and isolated cellsexpressing said CAR at their surface. The present invention also relatesto methods for engineering immune cells expressing 4G7-CAR at theirsurface which confers a prolonged “activated” state on the transducedcell. The present invention is particularly useful for the treatment ofB-cells lymphomas and leukemia.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

Novel specificities in T cells have been successfully generated throughthe genetic transfer of transgenic T cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T cells.CARs have successfully allowed T cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

CD19 is an attractive target for immunotherapy because the vast majorityof B-acute lymphoblastic leukemia (B-ALL) uniformly express CD19,whereas expression is absent on non hematopoietic cells, as well asmyeloid, erythroid, and T cells, and bone marrow stem cells. Clinicaltrials targeting CD19 on B-cell malignancies are underway withencouraging anti-tumor responses. Most infuse T cells geneticallymodified to express a chimeric antigen receptor (CAR) with specificityderived from the scFv region of a CD19-specific mouse monoclonalantibody FMC63 (Nicholson, Lenton et al. 1997; Cooper, Topp et al. 2003;Cooper, Jena et al. 2012) (International application: WO2013/126712).However, there is still a need to improve construction of CARs that showbetter compatibility with T-cell proliferation, in order to allow thecells expressing such CARs to reach significant clinical advantage.

SUMMARY OF THE INVENTION

The inventors have generated a CD19 specific CAR (4G7-CAR) comprising ascFV derived from the CD19 specific monoclonal antibody, 4G7, and havesurprisingly found that introduction of the resulting 4G7-CAR intoprimary T cells could confer a prolonged “activated” state on thetransduced cell independently of antigen binding. Following non-specificactivation in vitro (e.g. with anti CD3/CD28 coated beads andrecombinant IL2), these cells displayed an increased cell size (blastformation) as well as the expression of activation markers (CD25) overan extended time period compared to cells transduced with a similar CARcomprising the FMC63 scFV. This long-term activation permits extendedproliferation and provides an antigen-independent mechanism forexpansion of 4G7-CAR cells in vitro.

The present invention thus provides a chimeric antigen receptorcomprising at least one extracellular ligand binding domain, atransmembrane domain and at least one signal transducing domain, whereinsaid extracellular ligand binding domain comprises a scFV derived fromspecific monoclonal antibody, 4G7. In particular, the CAR of the presentinvention once transduced into an immune cell contributes to antigenindependent activation and proliferation of the cell. The presentinvention also relates to nucleic acid, vectors encoding the CARcomprising a scFV derived from the CD19 specific monoclonal antibody 4G7and methods of engineering immune cells comprising introducing into saidcell the 4G7 CAR. The present invention also relates to geneticallymodified immune cells expressing at their surface the 4G7, particularlyimmune cells which proliferate independently of antigen mechanism. Thegenetically modified immune cells of the present invention areparticularly useful for therapeutic applications such as B-cell lymphomaor leukemia treatments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Proliferation of TCR alpha inactivated T cells (KO) transducedwith 4G7-CAR lentiviral vector compared to non transduced KO T cells(NTD). Proliferation was followed during 30 days after (IL2+CD28) or not(IL2) a step of reactivation with soluble anti-CD28.

FIG. 2: CD25 activation marker expression analysis at the surface ofinactivated TCR alpha T cells transduced with 4G7-CAR lentiviral vector,gated on the basis of 4G7-CAR expression (CAR+, CAR−) and compared toCD25 expression on TCR alpha positive non electroporated (NEP) or TCRalpha disrupted but non tranduced (NTD) cells. CD25 expression wasanalyzed after (IL2+CD28) or not (IL2) a step of reactivation withsoluble anti-CD28.

FIG. 3: CAR expression analysis at the surface of T cells transducedwith a lentiviral vector encoding either the 4G7-CAR or the FMC63-CAR.The analysis was done 3, 8 and 15 days post transduction by flowcytometry. NT refers to no transduced T cells.

FIG. 4: CD25 expression analysis at the surface of T cells transducedwith a lentiviral vector encoding either the 4G7-CAR or the FMC63-CAR.The analysis was done 3, 8 and 15 days post transduction by flowcytometry. NT refers to no transduced T cells.

FIG. 5: Size analysis of T cells transduced with a lentiviral vectorencoding either the 4G7-CAR or the FMC63-CAR. The analysis was done 3, 8and 15 days post transduction by flow cytometry. NT refers to notransduced T cells.

FIG. 6: Proliferation of T cells transduced with 4G7-CAR compared toFMC63 lentiviral vector. Proliferation was followed during 20 days after(CD28) or not (−) a step of reactivation with soluble anti-CD28. NTDrefers to no transduced T cells.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, biochemistry, genetics, and molecularbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, NewYork: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis(M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; NucleicAcid Hybridization (B. D. Harries & S. J. Higgins eds. 1984);Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

CD19 Specific Chimeric Antigen Receptor

The present invention relates to a chimeric antigen receptor (CAR)comprising an extracellular ligand-binding domain, a transmembranedomain and a signaling transducing domain.

The term “extracellular ligand-binding domain” as used herein is definedas an oligo- or polypeptide that is capable of binding a ligand.Preferably, the domain will be capable of interacting with a cellsurface molecule. For example, the extracellular ligand-binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state.

In a preferred embodiment, said extracellular ligand-binding domaincomprises a single chain antibody fragment (scFv) comprising the light(V_(L)) and the heavy (V_(H)) variable fragment of a target antigenspecific monoclonal antibody joined by a flexible linker. In a preferredembodiment, said scFV is derived from the CD19 monoclonal antibody 4G7(Peipp, Saul et al. 2004), preferably said scFV of the present inventioncomprises a part of the CD19 monoclonal antibody 4G7 immunoglobulingamma 1 heavy chain (GenBank: CAD88275.1; SEQ ID NO: 1) and a part ofthe CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain(GenBank: CAD88204.1; SEQ ID NO: 2), preferably linked together by aflexible linker. In a preferred embodiment, said scFV of the presentinvention comprises the variable fragments of the CD19 monoclonalantibody 4G7 immunoglobulin gamma 1 heavy chain (SEQ ID NO: 3) and thevariable fragments of the CD19 monoclonal antibody 4G7 immunoglobulinkappa light chain (SEQ ID NO: 4 or SEQ ID NO: 5) linked together by aflexible linker. In particular embodiment said flexible linker has theamino acid sequence (SEQ ID NO: 6).

In other words, said CAR comprises an extracellular ligand-biding domainwhich comprises a single chain FV fragment derived from a CD19 specificmonoclonal antibody 4G7. In a particular embodiment, said scFV comprisesa part of amino acid sequences selected from the group consisting of:SEQ ID NO: 1 to 5. In a preferred embodiment said scFV comprises atleast 70%, preferably at least 80%, more preferably at least 90%, 95%97% or 99% sequence identity with amino acid sequence selected from thegroup consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

The signal transducing domain or intracellular signaling domain of theCAR according to the present invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the CAR is expressed. For example, the effectorfunction of a T cell can be a cytolytic activity or helper activityincluding the secretion of cytokines. Thus, the term “signal transducingdomain” refers to the portion of a protein which transduces the effectorsignal function signal and directs the cell to perform a specializedfunction.

Preferred examples of signal transducing domain for use in a CAR can bethe cytoplasmic sequences of the T cell receptor and co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement, as well as any derivate or variant of thesesequences and any synthetic sequence that has the same functionalcapability. Signal transduction domain comprises two distinct classes ofcytoplasmic signaling sequence, those that initiate antigen-dependentprimary activation, and those that act in an antigen-independent mannerto provide a secondary or co-stimulatory signal. Primary cytoplasmicsignaling sequence can comprise signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are welldefined signaling motifs found in the intracytoplasmic tail of a varietyof receptors that serve as binding sites for syk/zap70 class tyrosinekinases. Examples of ITAM used in the invention can include as nonlimiting examples those derived from TCRzeta, FcRgamma, FcRbeta,FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b andCD66d. In a preferred embodiment, the signaling transducing domain ofthe CAR can comprise the CD3zeta signaling domain which has amino acidsequence with at least 70%, preferably at least 80%, more preferably atleast 90%, 95% 97% or 99% sequence identity with amino acid sequenceselected from the group consisting of (SEQ ID NO: 10).

In particular embodiment the signal transduction domain of the CAR ofthe present invention comprises a co-stimulatory signal molecule. Aco-stimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient immuneresponse. “Co-stimulatory ligand” refers to a molecule on an antigenpresenting cell that specifically binds a cognate co-stimulatorymolecule on a T-cell, thereby providing a signal which, in addition tothe primary signal provided by, for instance, binding of a TCR/CD3complex with an MEW molecule loaded with peptide, mediates a T cellresponse, including, but not limited to, proliferation activation,differentiation and the like. A co-stimulatory ligand can include but isnot limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory igand (ICOS-L), intercellular adhesionmolecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibodythat binds Toll ligand receptor and a ligand that specifically bindswith B7-H3. A co-stimulatory ligand also encompasses, inter alia, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CO2,CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on aT-cell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and Toll ligand receptor.Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand thatspecifically binds with CD83 and the like.

In a preferred embodiment, the signal transduction domain of the CAR ofthe present invention comprises a part of co-stimulatory signal moleculeselected from the group consisting of fragment of 4-1BB (GenBank:AAA53133.) and CD28 (NP_006130.1). In particular the signal transductiondomain of the CAR of the present invention comprises amino acid sequencewhich comprises at least 70%, preferably at least 80%, more preferablyat least 90%, 95% 97% or 99% sequence identity with amino acid sequenceselected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.

The CAR according to the present invention is expressed on the surfacemembrane of the cell. Thus, the CAR can comprise a transmembrane domain.The distinguishing features of appropriate transmembrane domainscomprise the ability to be expressed at the surface of a cell,preferably in the present invention an immune cell, in particularlymphocyte cells or Natural killer (NK) cells, and to interact togetherfor directing cellular response of immune cell against a predefinedtarget cell. The transmembrane domain can be derived either from anatural or from a synthetic source. The transmembrane domain can bederived from any membrane-bound or transmembrane protein. As nonlimiting examples, the transmembrane polypeptide can be a subunit of theT cell receptor such as α, β, γ or δ, polypeptide constituting CD3complex, IL2 receptor p55 (α chain), p75 (β chain) or γ chain, subunitchain of Fc receptors, in particular Fcγ receptor III or CD proteins.Alternatively the transmembrane domain can be synthetic and can comprisepredominantly hydrophobic residues such as leucine and valine. In apreferred embodiment said transmembrane domain is derived from the humanCD8 alpha chain (e.g. NP_001139345.1). The transmembrane domain canfurther comprise a stalk region_between said extracellularligand-binding domain and said transmembrane domain. The term “stalkregion” used herein generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to the extracellularligand-binding domain. In particular, stalk region are used to providemore flexibility and accessibility for the extracellular ligand-bindingdomain. A stalk region may comprise up to 300 amino acids, preferably 10to 100 amino acids and most preferably 25 to 50 amino acids. Stalkregion may be derived from all or part of naturally occurring molecules,such as from all or part of the extracellular region of CD8, CD4 orCD28, or from all or part of an antibody constant region. Alternativelythe stalk region may be a synthetic sequence that corresponds to anaturally occurring stalk sequence, or may be an entirely syntheticstalk sequence. In a preferred embodiment said stalk region is a part ofhuman CD8 alpha chain (e.g. NP_001139345.1). In another particularembodiment, said transmembrane and hinge domains comprise a part ofhuman CD8 alpha chain, preferably which comprises at least 70%,preferably at least SO %, more preferably at least 90%, 95% 97% or 99%sequence identity with amino acid sequence selected from the groupconsisting of SEQ ID NO: 13.

In a particular embodiment, said Chimeric Antigen Receptor of thepresent invention comprises a scFV derived from the CD19 monoclonalantibody 4G7, a CDS alpha human hinge and transmembrane domain, the CD3zeta signaling domain and 4-1BB signaling domain. Preferably, the 4G7CAR of the present invention comprises at least 70%, preferably at least80%, more preferably at least 90%, 95% 97% or 99% sequence identity withamino acid sequence selected from the group consisting of SEQ ID NO: 14and 15.

Downregulation or mutation of target antigens is commonly observed incancer cells, creating antigen-loss escape variants. Thus, to offsettumor escape and render immune cell more specific to target, the CD19specific CAR can comprise another extracellular ligand-binding domains,to simultaneously bind different elements in target thereby augmentingimmune cell activation and function. In one embodiment, theextracellular ligand-binding domains can be placed in tandem on the sametransmembrane polypeptide, and optionally can be separated by a linker.In another embodiment, said different extracellular ligand-bindingdomains can be placed on different transmembrane polypeptides composingthe CAR. In another embodiment, the present invention relates to apopulation of CARs comprising each one different extracellular ligandbinding domains. In a particular, the present invention relates to amethod of engineering immune cells comprising providing an immune celland expressing at the surface of said cell a population of CAR each onecomprising different extracellular ligand binding domains. In anotherparticular embodiment, the present invention relates to a method ofengineering an immune cell comprising providing an immune cell andintroducing into said cell polynucleotides encoding polypeptidescomposing a population of CAR each one comprising differentextracellular ligand binding domains. By population of CARs, it is meantat least two, three, four, five, six or more CARs each one comprisingdifferent extracellular ligand binding domains. The differentextracellular ligand binding domains according to the present inventioncan preferably simultaneously bind different elements in target therebyaugmenting immune cell activation and function. The present inventionalso relates to an isolated immune cell which comprises a population ofCARs each one comprising different extracellular ligand binding domains.

Polynucleotides, Vectors:

The present invention also relates to polynucleotides, vectors encodingthe above described CAR according to the invention. In a preferredembodiment, the present invention relates to a polynucleotide comprisingthe nucleic acid sequence SEQ ID NO: 17. In a preferred embodiment, thepolynucleotide has at least 70%, preferably at least 80%, morepreferably at least 90%, 95% 97% or 99% sequence identity with nucleicacid sequence selected from the group consisting of SEQ ID NO: 17.

The polynucleotide may consist in an expression cassette or expressionvector (e.g. a plasmid for introduction into a bacterial host cell, or aviral vector such as a baculovirus vector for transfection of an insecthost cell, or a plasmid or viral vector such as a lentivirus fortransfection of a mammalian host cell).

In a particular embodiment, the different nucleic acid sequences can beincluded in one polynucleotide or vector which comprises a nucleic acidsequence encoding ribosomal skip sequence such as a sequence encoding a2A peptide. 2A peptides, which were identified in the Aphthovirussubgroup of picornaviruses, causes a ribosomal “skip” from one codon tothe next without the formation of a peptide bond between the two aminoacids encoded by the codons (see (Donnelly and Elliott 2001; Atkins,Wills et al. 2007; Doronina, Wu et al. 2008)). By “codon” is meant threenucleotides on an mRNA (or on the sense strand of a DNA molecule) thatare translated by a ribosome into one amino acid residue. Thus, twopolypeptides can be synthesized from a single, contiguous open readingframe within an mRNA when the polypeptides are separated by a 2Aoligopeptide sequence that is in frame. Such ribosomal skip mechanismsare well known in the art and are known to be used by several vectorsfor the expression of several proteins encoded by a single messengerRNA.

To direct, transmembrane polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in polynucleotide sequenceor vector sequence. The secretory signal sequence is operably linked tothe transmembrane nucleic acid sequence, i.e., the two sequences arejoined in the correct reading frame and positioned to direct the newlysynthesized polypeptide into the secretory pathway of the host cell.Secretory signal sequences are commonly positioned 5′ to the nucleicacid sequence encoding the polypeptide of interest, although certainsecretory signal sequences may be positioned elsewhere in the nucleicacid sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferredembodiment the signal peptide comprises the amino acid sequence SEQ IDNO: 18 and 19.

Those skilled in the art will recognize that, in view of the degeneracyof the genetic code, considerable sequence variation is possible amongthese polynucleotide molecules. Preferably, the nucleic acid sequencesof the present invention are codon-optimized for expression in mammaliancells, preferably for expression in human cells. Codon-optimizationrefers to the exchange in a sequence of interest of codons that aregenerally rare in highly expressed genes of a given species by codonsthat are generally frequent in highly expressed genes of such species,such codons encoding the amino acids as the codons that are beingexchanged.

In a preferred embodiment, the polynucleotide according to the presentinvention comprises the nucleic acid sequence selected from the groupconsisting of: SEQ ID NO: 17. The present invention relates topolynucleotides comprising a nucleic acid sequence that has at least70%, preferably at least 80%, more preferably at least 90%, 95% 97% or99% sequence identity with nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 17.

Methods of Engineering an Immune Cell:

In encompassed particular embodiment, the invention relates to a methodof preparing immune cells for immunotherapy comprising introducing intosaid immune cells the CAR according to the present invention andexpanding said cells. In particular embodiment, the invention relates toa method of engineering an immune cell comprising providing a cell andexpressing at the surface of said cell at least one CAR as describedabove. In particular embodiment, the method comprises transforming thecell with at least one polynucleotide encoding CAR as described above,and expressing said polynucleotides into said cell.

In a preferred embodiment, said polynucleotides are included inlentiviral vectors in view of being stably expressed in the cells.

In another embodiment, said method further comprises a step ofgenetically modifying said cell by inactivating at least one geneexpressing one component of the TCR, a target for an immunosuppressiveagent, HLA gene and/or an immune checkpoint gene such as PDCD1 orCTLA-4. In a preferred embodiment, said gene is selected from the groupconsisting of TCRalpha, TCRbeta, CD52, GR, PD1 and CTLA-4. In apreferred embodiment said method further comprises introducing into saidT cells a rare-cutting endonuclease able to selectively inactivate byDNA cleavage said genes. In a more preferred embodiment saidrare-cutting endonuclease is TALE-nuclease or Cas9 endonuclease.

Delivery Methods

The different methods described above involve introducing CAR into acell. As non-limiting example, said CAR can be introduced as transgenesencoded by one plasmidic vector. Said plasmid vector can also contain aselection marker which provides for identification and/or selection ofcells which received said vector.

Polypeptides may be synthesized in situ in the cell as a result of theintroduction of polynucleotides encoding said polypeptides into thecell. Alternatively, said polypeptides could be produced outside thecell and then introduced thereto. Methods for introducing apolynucleotide construct into cells are known in the art and includingas non limiting examples stable transformation methods wherein thepolynucleotide construct is integrated into the genome of the cell,transient transformation methods wherein the polynucleotide construct isnot integrated into the genome of the cell and virus mediated methods.Said polynucleotides may be introduced into a cell by for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomeand the like. For example, transient transformation methods include forexample microinjection, electroporation or particle bombardment. Saidpolynucleotides may be included in vectors, more particularly plasmidsor virus, in view of being expressed in cells.

Engineered Immune Cells

The present invention also relates to isolated cells or cell linessusceptible to be obtained by said method to engineer cells. Inparticular said isolated cell comprises at least one CAR as describedabove. In another embodiment, said isolated cell comprises a populationof CARs each one comprising different extracellular ligand bindingdomains. In particular, said isolated cell comprises exogenouspolynucleotide sequence encoding CAR. Genetically modified immune cellsof the present invention are activated and proliferate independently ofantigen binding mechanisms.

In the scope of the present invention is also encompassed an isolatedimmune cell, preferably a T-cell obtained according to any one of themethods previously described. Said immune cell refers to a cell ofhematopoietic origin functionally involved in the initiation and/orexecution of innate and/or adaptative immune response. Said immune cellaccording to the present invention can be derived from a stem cell. Thestem cells can be adult stem cells, non-human embryonic stem cells, moreparticularly non-human stem cells, cord blood stem cells, progenitorcells, bone marrow stem cells, induced pluripotent stem cells,totipotent stem cells or hematopoietic stem cells. Representative humancells are CD34+ cells. Said isolated cell can also be a dendritic cell,killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cellselected from the group consisting of inflammatory T-lymphocytes,cytotoxic T-lymphocytes, regulatory T-lymphocytes or helperT-lymphocytes. In another embodiment, said cell can be derived from thegroup consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. Prior toexpansion and genetic modification of the cells of the invention, asource of cells can be obtained from a subject through a variety ofnon-limiting methods. Cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell can be derived from a healthy donor, from apatient diagnosed with cancer or from a patient diagnosed with aninfection. In another embodiment, said cell is part of a mixedpopulation of cells which present different phenotypic characteristics.In the scope of the present invention is also encompassed a cell lineobtained from a transformed T-cell according to the method previouslydescribed. Modified cells resistant to an immunosuppressive treatmentand susceptible to be obtained by the previous method are encompassed inthe scope of the present invention.

In another embodiment, said isolated cell according to the presentinvention comprises a polynucleotide encoding CAR.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells, even ifthe genetically modified immune cells of the present invention areactivated and proliferate independently of antigen binding mechanisms,the immune cells, particularly T-cells of the present invention can befurther activated and expanded generally using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869;7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; andU.S. Patent Application Publication No. 20060121005. T cells can beexpanded in vitro or in vivo.

Generally, the T cells of the invention are expanded by contact with anagent that stimulates a CD3 TCR complex and a co-stimulatory molecule onthe surface of the T cells to create an activation signal for theT-cell.

For example, chemicals such as calcium ionophore A23187, phorbol12-myristate 13-acetate (PMA), or mitogenic lectins likephytohemagglutinin (PHA) can be used to create an activation signal forthe T-cell.

As non limiting examples, T cell populations may be stimulated in vitrosuch as by contact with an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, a population of T cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza))that may contain factors necessary for proliferation and viability,including serum (e.g., fetal bovine or human serum), interleukin-2(IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, -10, -2, IL-15, TGFp, andTNF- or any other additives for the growth of cells known to the skilledartisan. Other additives for the growth of cells include, but are notlimited to, surfactant, plasmanate, and reducing agents such asN-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640,A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, withadded amino acids, sodium pyruvate, and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of T cells. Antibiotics, e.g., penicillin andstreptomycin, are included only in experimental cultures, not incultures of cells that are to be infused into a subject. The targetcells are maintained under conditions necessary to support growth, forexample, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO2). T cells that have been exposed to varied stimulationtimes may exhibit different characteristics

In another particular embodiment, said cells can be expanded byco-culturing with tissue or cells. Said cells can also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Therapeutic Applications

In another embodiment, isolated cell obtained by the different methodsor cell line derived from said isolated cell as previously described canbe used as a medicament. In another embodiment, said medicament can beused for treating cancer, particularly for the treatment of B-celllymphomas and leukemia in a patient in need thereof. In anotherembodiment, said isolated cell according to the invention or cell linederived from said isolated cell can be used in the manufacture of amedicament for treatment of a cancer in a patient in need thereof.

In another aspect, the present invention relies on methods for treatingpatients in need thereof, said method comprising at least one of thefollowing steps:

(a) providing an immune-cell obtainable by any one of the methodspreviously described;

(b) Administrating said transformed immune cells to said patient,

On one embodiment, said T cells of the invention can undergo robust invivo T cell expansion and can persist for an extended amount of time.

Said treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogeneicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

Cells that can be used with the disclosed methods are described in theprevious section. Said treatment can be used to treat patients diagnosedwith cancer. Cancers that may be treated may comprise nonsolid tumors(such as hematological tumors, including but not limited to pre-B ALL(pedriatic indication), adult ALL, mantle cell lymphoma, diffuse largeB-cell lymphoma and the like. Types of cancers to be treated with theCARs of the invention include, but are not limited to certain leukemiaor lymphoid malignancies. Adult tumors/cancers and pediatrictumors/cancers are also included.

It can be a treatment in combination with one or more therapies againstcancer selected from the group of antibodies therapy, chemotherapy,cytokines therapy, dendritic cell therapy, gene therapy, hormonetherapy, laser light therapy and radiation therapy.

According to a preferred embodiment of the invention, said treatment canbe administrated into patients undergoing an immunosuppressivetreatment. Indeed, the present invention preferably relies on cells orpopulation of cells, which have been made resistant to at least oneimmunosuppressive agent due to the inactivation of a gene encoding areceptor for such immunosuppressive agent. In this aspect, theimmunosuppressive treatment should help the selection and expansion ofthe T-cells according to the invention within the patient.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermaliy, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In another embodiment, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

In certain embodiments of the present invention, cells are administeredto a patient in conjunction with (e.g., before, simultaneously orfollowing) any number of relevant treatment modalities, including butnot limited to treatment with agents such as antiviral therapy,cidofovir and interleukin-2, Cytarabine (also known as ARA-C) ornataliziimab treatment for MS patients or efaliztimab treatment forpsoriasis patients or other treatments for PML patients. In furtherembodiments, the T cells of the invention may be used in combinationwith chemotherapy, radiation, immunosuppressive agents, such ascyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992;Bierer, Hollander et al. 1993). In a further embodiment, the cellcompositions of the present invention are administered to a patient inconjunction with (e.g., before, simultaneously or following) bone marrowtransplantation, T cell ablative therapy using either chemotherapyagents such as, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In anotherembodiment, the cell compositions of the present invention areadministered following B-cell ablative therapy such as agents that reactwith CD20, e.g., Rituxan. For example, in one embodiment, subjects mayundergo standard treatment with high dose chemotherapy followed byperipheral blood stem cell transplantation. In certain embodiments,following the transplant, subjects receive an infusion of the expandedimmune cells of the present invention. In an additional embodiment,expanded cells are administered before or following surgery.

Other Definitions

-   -   Unless otherwise specified, “a,” “an,” “the,” and “at least one”        are used interchangeably and mean one or more than one.—Amino        acid residues in a polypeptide sequence are designated herein        according to the one-letter code, in which, for example, Q means        Gln or Glutamine residue, R means Arg or Arginine residue and D        means Asp or Aspartic acid residue.    -   Amino acid substitution means the replacement of one amino acid        residue with another, for instance the replacement of an        Arginine residue with a Glutamine residue in a peptide sequence        is an amino acid substitution.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: a is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        tor c, h represents a, t or c, and n represents g, a, t or c.    -   “As used herein, “nucleic acid” or “polynucleotides” refers to        nucleotides and/or polynucleotides, such as deoxyribonucleic        acid (DNA) or ribonucleic acid (RNA), oligonucleotides,        fragments generated by the polymerase chain reaction (PCR), and        fragments generated by any of ligation, scission, endonuclease        action, and exonuclease action. Nucleic acid molecules can be        composed of monomers that are naturally-occurring nucleotides        (such as DNA and RNA), or analogs of naturally-occurring        nucleotides (e.g., enantiomeric forms of naturally-occurring        nucleotides), or a combination of both. Modified nucleotides can        have alterations in sugar moieties and/or in pyrimidine or        purine base moieties. Sugar modifications include, for example,        replacement of one or more hydroxyl groups with halogens, alkyl        groups, amines, and azido groups, or sugars can be        functionalized as ethers or esters. Moreover, the entire sugar        moiety can be replaced with sterically and electronically        similar structures, such as aza-sugars and carbocyclic sugar        analogs. Examples of modifications in a base moiety include        alkylated purines and pyrimidines, acylated purines or        pyrimidines, or other well-known heterocyclic substitutes.        Nucleic acid monomers can be linked by phosphodiester bonds or        analogs of such linkages. Nucleic acids can be either single        stranded or double stranded.    -   By chimeric antigen receptor (CAR) is intended molecules that        combine a binding domain against a component present on the        target cell, for example an anti body-based specificity for a        desired antigen (e.g., tumor antigen) with a T cell        receptor-activating intracellular domain to generate a chimeric        protein that exhibits a specific anti-target cellular immune        activity. Generally, CAR consists of an extracellular single        chain antibody (scFvFc) fused to the intracellular signaling        domain of the T cell antigen receptor complex zeta chain        (scFvFcζ) and have the ability, when expressed in T cells, to        redirect antigen recognition based on the monoclonal antibody's        specificity. One example of CAR used in the present invention is        a CAR directing against CD19 antigen and can comprise as non        limiting example the amino acid sequence: SEQ ID NO: 14.    -   The term “endonuclease” refers to any wild-type or variant        enzyme capable of catalyzing the hydrolysis (cleavage) of bonds        between nucleic acids within a DNA or RNA molecule, preferably a        DNA molecule. Endonucleases do not cleave the DNA or RNA        molecule irrespective of its sequence, but recognize and cleave        the DNA or RNA molecule at specific polynucleotide sequences,        further referred to as “target sequences” or “target sites”.        Endonucleases can be classified as rare-cutting endonucleases        when having typically a polynucleotide recognition site greater        than 12 base pairs (bp) in length, more preferably of 14-55 bp.        Rare-cutting endonucleases significantly increase HR by inducing        DNA double-strand breaks (DSBs) at a defined locus (Perrin,        Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et        al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases        can for example be a homing endonuclease (Paques and Duchateau        2007), a chimeric Zinc-Finger nuclease (ZFN) resulting from the        fusion of engineered zinc-finger domains with the catalytic        domain of a restriction enzyme such as Fokl (Porteus and Carroll        2005), a Cas9 endonuclease from CRISPR system (Gasiunas,        Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran        et al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease        (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006).        In chemical endonucleases, a chemical or peptidic cleaver is        conjugated either to a polymer of nucleic acids or to another        DNA recognizing a specific target sequence, thereby targeting        the cleavage activity to a specific sequence. Chemical        endonucleases also encompass synthetic nucleases like conjugates        of orthophenanthroline, a DNA cleaving molecule, and        triplex-forming oligonucleotides (TFOs), known to bind specific        DNA sequences (Kalish and Glazer 2005). Such chemical        endonucleases are comprised in the term “endonuclease” according        to the present invention.    -   By a “TALE-nuclease” (TALEN) is intended a fusion protein        consisting of a nucleic acid-binding domain typically derived        from a Transcription Activator Like Effector (TALE) and one        nuclease catalytic domain to cleave a nucleic acid target        sequence. The catalytic domain is preferably a nuclease domain        and more preferably a domain having endonuclease activity, like        for instance I-TevI, CoIE7, NucA and Fok-I. In a particular        embodiment, the TALE domain can be fused to a meganuclease like        for instance I-Crel and I-Onul or functional variant thereof. In        a more preferred embodiment, said nuclease is a monomeric        TALE-Nuclease. A monomeric TALE-Nuclease is a TALE-Nuclease that        does not require dimerization for specific recognition and        cleavage, such as the fusions of engineered TAL repeats with the        catalytic domain of I-Tevl described in WO2012138927.        Transcription Activator like Effector (TALE) are proteins from        the bacterial species Xanthomonas comprise a plurality of        repeated sequences, each repeat comprising di-residues in        position 12 and 13 (RVD) that are specific to each nucleotide        base of the nucleic acid targeted sequence. Binding domains with        similar modular base-per-base nucleic acid binding properties        (MBBBD) can also be derived from new modular proteins recently        discovered by the applicant in a different bacterial species.        The new modular proteins have the advantage of displaying more        sequence variability than TAL repeats. Preferably, RVDs        associated with recognition of the different nucleotides are HD        for recognizing C, NG for recognizing T, NI for recognizing A,        NN for recognizing G or A, NS for recognizing A, C, G or T, HG        for recognizing T, IG for recognizing T, NK for recognizing G,        HA for recognizing C, ND for recognizing C, HI for recognizing        C, HN for recognizing G, NA for recognizing G, SN for        recognizing G or A and YG for recognizing T, TL for recognizing        A, VT for recognizing A or G and SW for recognizing A. In        another embodiment, critical amino acids 12 and 13 can be        mutated towards other amino acid residues in order to modulate        their specificity towards nucleotides A, T, C and G and in        particular to enhance this specificity. TALE-nuclease have been        already described and used to stimulate gene targeting and gene        modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove        2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011).        Engineered TAL-nucleases are commercially available under the        trade name TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013        Paris, France).

The rare-cutting endonuclease according to the present invention canalso be a Cas9 endonuclease. Recently, a new genome engineering tool hasbeen developed based on the RNA-guided Cas9 nuclease (Gasiunas,Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al.2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR(Clustered Regularly Interspaced Short palindromic Repeats) adaptiveimmune system (see for review (Sorek, Lawrence et al. 2013)). The CRISPRAssociated (Cas) system was first discovered in bacteria and functionsas a defense against foreign DNA, either viral or plasmid.CRISPR-mediated genome engineering first proceeds by the selection oftarget sequence often flanked by a short sequence motif, referred as theprotospacer adjacent motif (PAM). Following target sequence selection, aspecific crRNA, complementary to this target sequence is engineered.Trans-activating crRNA (tracrRNA) required in the CRISPR type II systemspaired to the crRNA and bound to the provided Cas9 protein. Cas9 acts asa molecular anchor facilitating the base pairing of tracRNA with cRNA(Deltcheva, Chylinski et al. 2011). In this ternary complex, the dualtracrRNA: crRNA structure acts as guide RNA that directs theendonuclease Cas9 to the cognate target sequence. Target recognition bythe Cas9-tracrRNA:crRNA complex is initiated by scanning the targetsequence for homology between the target sequence and the crRNA. Inaddition to the target sequence-crRNA complementarity, DNA targetingrequires the presence of a short motif adjacent to the protospacer(protospacer adjacent motif—PAM). Following pairing between the dual-RNAand the target sequence, Cas9 subsequently introduces a blunt doublestrand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al.2010).

Rare-cutting endonuclease can be a homing endonuclease, also known underthe name of meganuclease. Such homing endonucleases are well-known tothe art (Stoddard 2005). Homing endonucleases recognize a DNA targetsequence and generate a single- or double-strand break. Homingendonucleases are highly specific, recognizing DNA target sites rangingfrom 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40bp in length. The homing endonuclease according to the invention may forexample correspond to a LAGLIDADG endonuclease, to a HNH endonuclease,or to a GIY-YIG endonuclease. Preferred homing endonuclease according tothe present invention can be an I-Crel variant.

-   -   By “delivery vector” or “delivery vectors” is intended any        delivery vector which can be used in the present invention to        put into cell contact (i.e “contacting”) or deliver inside cells        or subcellular compartments (i.e “introducing”) agents/chemicals        and molecules (proteins or nucleic acids) needed in the present        invention. It includes, but is not limited to liposomal delivery        vectors, viral delivery vectors, drug delivery vectors, chemical        carriers, polymeric carriers, lipoplexes, polyplexes,        dendrimers, microbubbles (ultrasound contrast agents),        nanoparticles, emulsions or other appropriate transfer vectors.        These delivery vectors allow delivery of molecules, chemicals,        macromolecules (genes, proteins), or other vectors such as        plasmids, peptides developed by Diatos. In these cases, delivery        vectors are molecule carriers. By “delivery vector” or “delivery        vectors” is also intended delivery methods to perform        transfection.    -   The terms “vector” or “vectors” refer to a nucleic acid molecule        capable of transporting another nucleic acid to which it has        been linked. A “vector” in the present invention includes, but        is not limited to, a viral vector, a plasmid, a RNA vector or a        linear or circular DNA or RNA molecule which may consists of a        chromosomal, non chromosomal, semi-synthetic or synthetic        nucleic acids. Preferred vectors are those capable of autonomous        replication (episomal vector) and/or expression of nucleic acids        to which they are linked (expression vectors). Large numbers of        suitable vectors are known to those of skill in the art and        commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e.g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies andvesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai),positive strand RNA viruses such as picornavirus and alphavirus, anddouble-stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include: avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996).

-   -   By “lentiviral vector” is meant HIV-Based lentiviral vectors        that are very promising for gene delivery because of their        relatively large packaging capacity, reduced immunogenicity and        their ability to stably transduce with high efficiency a large        range of different cell types. Lentiviral vectors are usually        generated following transient transfection of three (packaging,        envelope and transfer) or more plasmids into producer cells.        Like HIV, lentiviral vectors enter the target cell through the        interaction of viral surface glycoproteins with receptors on the        cell surface. On entry, the viral RNA undergoes reverse        transcription, which is mediated by the viral reverse        transcriptase complex. The product of reverse transcription is a        double-stranded linear viral DNA, which is the substrate for        viral integration in the DNA of infected cells. By “integrative        lentiviral vectors (or LV)”, is meant such vectors as        nonlimiting example, that are able to integrate the genome of a        target cell. At the opposite by “non-integrative lentiviral        vectors (or NILV)” is meant efficient gene delivery vectors that        do not integrate the genome of a target cell through the action        of the virus integrase.    -   Delivery vectors and vectors can be associated or combined with        any cellular permeabilization techniques such as sonoporation or        electroporation or derivatives of these techniques.    -   By cell or cells is int ended any eukaryotic living cells,        primary cells and cell lines derived from these organisms for in        vitro cultures.    -   By “primary cell” or “primary cells” are intended cells taken        directly from living tissue (i.e. biopsy material) and        established for growth in vitro, that have undergone very few        population doublings and are therefore more representative of        the main functional components and characteristics of tissues        from which they are derived from, in comparison to continuous        tumorigenic or artificially immortalized cell lines.

As non limiting examples cell lines can be selected from the groupconsisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells,U-937 cells; MRCS cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLacells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4cells.

All these cell lines can be modified by the method of the presentinvention to provide cell line models to produce, express, quantify,detect, study a gene or a protein of interest; these models can also beused to screen biologically active molecules of interest in research andproduction and various fields such as chemical, biofuels, therapeuticsand agronomy as non-limiting examples.

-   -   by “mutation” is intended the substitution, deletion, insertion        of up to one, two, three, four, five, six, seven, eight, nine,        ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty        five, thirty, forty, fifty, or more nucleotides/amino acids in a        polynucleotide (cDNA, gene) or a polypeptide sequence. The        mutation can affect the coding sequence of a gene or its        regulatory sequence. It may also affect the structure of the        genomic sequence or the structure/stability of the encoded mRNA.    -   by “variant(s)”, it is intended a repeat variant, a variant, a        DNA binding variant, a TALE-nuclease variant, a polypeptide        variant obtained by mutation or replacement of at least one        residue in the amino acid sequence of the parent molecule.    -   by “functional variant” is intended a catalytically active        mutant of a protein or a protein domain; such mutant may have        the same activity compared to its parent protein or protein        domain or additional properties, or higher or lower activity.    -   “identity” refers to sequence identity between two nucleic acid        molecules or polypeptides. Identity can be determined by        comparing a position in each sequence which may be aligned for        purposes of comparison. When a position in the compared sequence        is occupied by the same base, then the molecules are identical        at that position. A degree of similarity or identity between        nucleic acid or amino acid sequences is a function of the number        of identical or matching nucleotides at positions shared by the        nucleic acid sequences. Various alignment algorithms and/or        programs may be used to calculate the identity between two        sequences, including FASTA, or BLAST which are available as a        part of the GCG sequence analysis package (University of        Wisconsin, Madison, Wis.), and can be used with e.g., default        setting. For example, polypeptides having at least 70%, 85%,        90%, 95%, 98% or 99% identity to specific polypeptides described        herein and preferably exhibiting substantially the same        functions, as well as polynucleotide encoding such polypeptides,        are contemplated.    -   “similarity” describes the relationship between the amino acid        sequences of two or more polypeptides. BLASTP may also be used        to identify an amino acid sequence having at least 70%, 75%,        80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence        similarity to a reference amino acid sequence using a similarity        matrix such as BLOSUM45, BLOSUM62 or BLOSUM80. Unless otherwise        indicated a similarity score will be based on use of BLOSUM62.        When BLASTP is used, the percent similarity is based on the        BLASTP positives score and the percent sequence identity is        based on the BLASTP identities score. BLASTP “Identities” shows        the number and fraction of total residues in the high scoring        sequence pairs which are identical; and BLASTP “Positives” shows        the number and fraction of residues for which the alignment        scores have positive values and which are similar to each other.        Amino acid sequences having these degrees of identity or        similarity or any intermediate degree of identity of similarity        to the amino acid sequences disclosed herein are contemplated        and encompassed by this disclosure. The polynucleotide sequences        of similar polypeptides are deduced using the genetic code and        may be obtained by conventional means. A polynucleotide encoding        such a functional variant would be produced by reverse        translating its amino acid sequence using the genetic code.    -   “signal-transducing domain” or “co-stimulatory ligand” refers to        a molecule on an antigen presenting cell that specifically binds        a cognate co-stimulatory molecule on a T-cell, thereby providing        a signal which, in addition to the primary signal provided by,        for instance, binding of a TCR/CD3 complex with an MHC molecule        loaded with peptide, mediates a T cell response, including, but        not limited to, proliferation activation, differentiation and        the like. A co-stimulatory ligand can include but is not limited        to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,        inducible costimulatory igand (ICOS-L), intercellular adhesion        molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB,        HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist        or antibody that binds Toll ligand receptor and a ligand that        specifically binds with B7-H3. A co-stimulatory ligand also        encompasses, inter alia, an antibody that specifically binds        with a co-stimulatory molecule present on a T cell, such as but        not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,        lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,        LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on aTcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and Toll ligand receptor.

A “co-stimulatory signal” as used herein refers to a signal, which incombination with primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

-   -   The term “extracellular ligand-binding domain” as used herein is        defined as an oligo- or polypeptide that is capable of binding a        ligand. Preferably, the domain will be capable of interacting        with a cell surface molecule. For example, the extracellular        ligand-binding domain may be chosen to recognize a ligand that        acts as a cell surface marker on target cells associated with a        particular disease state. Thus examples of cell surface markers        that may act as ligands include those associated with viral,        bacterial and parasitic infections, autoimmune disease and        cancer cells.

The term “subject” or “patient” as used herein includes all members ofthe animal kingdom including non-human primates and humans.

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES Example 1: Proliferation of TCRalpha Inactivated CellsExpressing a 4G7-CAR

Heterodimeric TALE-nuclease targeting two 17-bp long sequences (calledhalf targets) separated by an 15-bp spacer within T-cell receptor alphaconstant chain region (TRAC) gene were designed and produced. Each halftarget is recognized by repeats of the half TALE-nucleases listed inTable 1.

Target Repeat Half TALE- Target sequence sequence nuclease TRAC_T01TTGTCCCAC Repeat TRAC_T01-L AGATATCC TRAC_T01-L TALEN Agaaccctg(SEQ ID NO: (SEQ ID NO: accctg 21) 23) CCGTGTACC Repeat TRAC_T01-RAGCTGAGA TRAC_T01-R TALEN (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 20) 22)24)

Each TALE-nuclease construct was subcloned using restriction enzymedigestion in a mammalian expression vector under the control of the T7promoter. mRNA encoding TALE-nuclease cleaving TRAC genomic sequencewere synthesized from plasmid carrying the coding sequence downstreamfrom the T7 promoter.

Purified T cells preactivated during 72 hours with antiCD3/CD28 coatedbeads were transfected with each of the 2 mRNAs encoding both halfTRAC_T01 TALE-nucleases. 48 hours post-transfection, T cells weretransduced with a lentiviral vector encoding 4G7-CAR (SEQ ID NO: 14). 2days post-transduction, CD3NEG cells were purified using anti-CD3magnetic beads and 5 days post-transduction cells were reactivated withsoluble anti-CD28 (5 μg/ml).

Cell proliferation was followed for up to 30 days after reactivation bycounting cell 2 times per week. The FIG. 1 shows the fold induction incell number respect to the amount of cells present at day 2 postreactivation for two different donors. Increased proliferation in TCRalpha inactivated cells expressing the 4G7-CAR, especially whenreactivated with anti-CD28, was observed compared to non transducedcells.

To investigate whether the human T cells expressing the 4G7-CAR displayactivated state, the expression of the activation marker CD25 wasanalyzed by FACS 7 days post transduction. As indicated in FIG. 2,purified cells transduced with the lentiviral vector encoding 4G7-CARexpressed considerably more CD25 at their surface than the nontransduced cells. Increased CD25 expression is observed both in CD28reactivation or no reactivation conditions.

Example 2: Comparison of Basal Activation of Primary Human T CellsExpressing the 4G7-CAR and the Classical FMC63-CAR

To determine whether 4G7 scFV confers a prolonged “activated” state onthe transduced cell, basal activation of T cell transduced with CARharboring a 4G7 scFV (SEQ ID NO: 17 encoded SEQ ID NO: 15) or aclassical FMC63 scFV (SEQ ID NO: 16) was compared.

Purified human T cells were transduced according to the followingprotocol: briefly, 1×10⁶ CD3+ cells preactivated during 3 days with antiCD3/CD28 coated beads and recombinant IL2 were transduced withlentiviral vectors encoding the 4G7-CAR (SEQ ID NO: 15) and theFMC63-CAR (SEQ ID NO: 16) at an MOI of 5 in 12-well non tissue cultureplates coated with 30 μg/ml retronectin. 24 hours post transduction themedium was removed and replaced by fresh medium. The cells were thenmaintained at a concentration of 1×10⁶ cells/ml throughout the cultureperiod by cell enumeration every 2-3 days.

3, 8 and 15 days post transduction with the lentiviral vector encodingeither the 4G7-CAR or the FMC63-CAR, the percentage of CAR expressingcells was assessed by flow cytometry. It was observed that theefficiency of transduction was relatively equivalent with the twolentiviral vectors FIG. 3.

It was then investigated whether the human T cells expressing the4G7-CAR exhibited a more activated state than the human T cellsexpressing the FMC63-CAR. For that purpose the expression of theactivation marker CD25 was compared at the surface of T cells transducedwith the 2 lentiviral vectors at different time points. As indicated inthe FIGS. 4, 3 and 8 days post transduction, the cells transduced withthe lentiviral vector encoding the 4G7-CAR expressed considerably moreCD25 at their surface than the cells transduced with the lentiviralvector encoding the FMC63-CAR.

The size of the 4G7-CAR or FMC63-CAR transduced cells was also assessedby flow cytometry at different time points. It was observed that thecells expressing the 4G7-CAR were bigger than the cells expressing theFMC63-CAR 3, 8 and 15 days post transduction FIG. 5.

Following non-specific activation in vitro, 4G7-CAR transduced cellsdisplay an increased cell size (blast formation) as well as theexpression of activation markers (CD25) over an extended time period.This long-term activation permits extended proliferation compared tocells transduced with a similar CAR containing the FMC63 ScFv.

Example 3: Comparison of Proliferation of Primary Human T CellsExpressing the 4G7-CAR and the Classical FMC63-CAR

To determine whether 4G7 scFV confers a higher proliferation activity,proliferation of T cell transduced with CAR harboring a 4G7 scFV (SEQ IDNO: 17 encoded SEQ ID NO: 15) or a classical FMC63 scFV (SEQ ID NO: 16)was followed up to 20 days by counting cell two times per week. Purifiedhuman T cells were transduced according to the following protocol:briefly, 1×10⁶ CD3+ cells preactivated during 3 days with anti CD3/CD28coated beads and recombinant IL2 were transduced with lentiviral vectorsencoding the 4G7-CAR (SEQ ID NO: 15) and the FMC63-CAR (SEQ ID NO: 16).The cells were then maintained under classical conditions and werereactivated at Day 12. Cells were seeded at the same density and werecounted two times per week during 20 days. As represented in FIG. 6,proliferation activity of T-cells expressing the 4G7-CAR is twofoldhigher compared to those of cells expressing the classical FMC63-CAR.

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What is claimed is:
 1. A method of treating cancer, the method comprising administering to a subject having a cancer expressing CD19 a genetically modified T cell expressing a chimeric antigen receptor (CAR) comprising, in order from N-terminus to C-terminus: i) the amino acid sequence of SEQ ID NO: 7; ii) the amino acid sequence of SEQ ID NO: 13; iii) the amino acid sequence of SEQ ID NO: 11; and iv) the amino acid sequence of SEQ ID NO:
 10. 2. The method of claim 1, wherein the cancer is a non-solid tumor.
 3. The method of claim 1, wherein the cancer is a B-cell lymphoma or leukemia.
 4. The method of claim 1, wherein the genetically modified T cell administered to the subject is an allogeneic T cell.
 5. The method of claim 1, wherein the genetically modified T cell administered to the subject is engineered from a cell of a healthy donor.
 6. The method of claim 1, wherein the genetically modified T cell administered to the subject is a cytotoxic T lymphocyte (CTL).
 7. The method of claim 1, wherein 10⁴-10⁹ T cells per kg body weight are administered to the subject.
 8. The method of claim 1, wherein the genetically modified T cell is administered intravenously to the subject.
 9. The method of claim 1, comprising administering alemtuzumab to the subject before, during, and/or after administration of the genetically modified T cell.
 10. The method of claim 1, wherein the CAR further comprises the amino acid sequence of SEQ ID NO: 18 at the N-terminus.
 11. The method of claim 1, wherein the CAR has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:
 15. 12. The method of claim 11, wherein the CAR has at least 99% sequence identity with the amino acid sequence of SEQ ID NO:
 15. 13. A method of treating cancer, the method comprising administering to a subject having a cancer expressing CD19 a genetically modified T cell expressing a chimeric antigen receptor (CAR), wherein the CAR has at least 99% sequence identity with the amino acid sequence of SEQ ID NO:
 15. 14. The method of claim 13, wherein the cancer is a B-cell lymphoma or leukemia.
 15. The method of claim 13, wherein the genetically modified T cell administered to the subject is an allogeneic T cell.
 16. The method of claim 13, wherein the genetically modified T cell is administered intravenously to the subject.
 17. The method of claim 13, comprising administering alemtuzumab to the subject before, during, and/or after administration of the genetically modified T cell. 